PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is a progressive disorder that begins long before clinical symptoms appear. Prior research has found that the asymptomatic (no symptoms, ASYM) phase of AD is linked to abnormal brain accumulations of the protein fragment beta-amyloid and the protein tau in the brain. Yet, current studies suggest that other mechanisms are involved in initiating the disease process. In our preliminary studies, we analyzed the protein glycosylation of the dorsolateral prefrontal cortex from ASYM and AD post-mortem brains. Based on initial findings that there are significant changes in glycosylation by looking at O-GlcNAc, N- and O- glycans in ASYM versus AD samples, we hypothesize that the glycomes, including the glyco(phospho)proteins and their associated O-GlcNAc, O-GalNAc-glycans, and N-glycans, are different in ASYM versus AD patient samples, despite many similarities in overall histopathology. To address this hypothesis, we will analyze the phosphoproteomes, glycophosphoproteome, glycoproteome, and the transcriptomes responsible for these differences in 125 control brain specimens, 125 ASYM brain specimens, and 125 clinical AD patient specimens over this 5 year study. Our proposal leverages a novel suite of resources and technologies, including unique brain tissues, glyco(phospho)proteomics, glycomics, and bioinformatics. We have developed a robust workflow to process and analyze such samples, including the use of novel synthetic glycans and glycopeptides with all types of glycosylation that also incorporate phosphorylation features to provide quantitative information and serve as standards for mass spectrometry methods. We propose the following 4 specific aims. Aim 1: We will define the O-GlcNAc-glycoproteome and O-phospho-proteomes on intracellular glycoproteins in control, ASYM, and AD patient samples. Aim 2: We will define the N-glycoproteome which occurs mainly on extracellular and secreted glycoproteins and define the glycan structures and sites of modification. Aim 3: We will define the O-GalNAc-glycoproteome which occurs mainly on extracellular and secreted glycoproteins and define glycan structures and sites of modification. Aim 4: We will integrate our new data on the glycome from the control, ASYM, and AD specimens with other `omics data generated in the AMP-AD Consortium, to relate the expression of glycan-related genes to changes in the glycomes. Our findings will provide high impact, new information to help identify novel biomarkers for AD and distinguish the glycophosphoproteomes of AD from asymptomatic brains. These data will also generate a usable and accessible database for researchers interested in AD brain glycomics.